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首页> 美国卫生研究院文献>Molecular and Cellular Biology >Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.
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Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

机译：复制缺陷的嵌合辅助原病毒和影响感受态病毒产生的因素：通过金属硫蛋白启动子表达莫洛尼鼠白血病病毒结构基因。

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Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.
机译：通过用小鼠金属硫蛋白I启动子替换5'长末端重复序列和相邻的前病毒序列，从嗜温莫洛尼鼠白血病病毒的前病毒衍生出两种嵌合辅助原病毒。这些嵌合原病毒中的一种被设计为表达病毒的gag-pol基因，而另一种仅被设计为表达env基因。当转染到NIH 3T3细胞中时，这些辅助原病毒无法产生有效的病毒，但确实表达了组装感染性载体所需的Zn2 +诱导的反式病毒功能。一种辅助细胞系（克隆32）以与转染相同载体的Psi-2和PA317细胞系所支持的水平相当的水平支持载体装配。携带新霉素磷酸转移酶基因且与嵌合辅助原病毒的5'端缺乏重叠序列同源性的有缺陷的原病毒可以被转染到辅助细胞系中，而不会产生具有复制能力的病毒。转染的辅助细胞的大规模培养产生的滴度约为10（4）G418r CFU / ml，而单个克隆产生的滴度在0至2.6 X 10（4）CFU / ml之间。相反，与嵌合辅助原病毒的5'末端共享同源重叠病毒序列的有缺陷的原病毒在转染到辅助细胞系中时容易产生感染性病毒。从辅助原病毒中删除多个顺式作用功能，并消除在辅助原病毒和载体原病毒5'端重叠的序列同源性，都有助于提高该系统的遗传稳定性。

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